Method for growing and metabolizing microbes

ABSTRACT

The present invention is generally directed to a method for releasing trapped oil in reservoirs. The method hereof includes identifying a reservoir, obtaining a microbial community sample of the reservoir, maintaining the sample under high temperature and high pressure conditions that mimic natural conditions of the reservoir, growing the sample on at least one substrate, determining a targeted treatment regime for the reservoir based on the positive growth of the sample on the substrate, injecting the reservoir with the targeted substrate to release trapped oil in the reservoir, monitoring the reservoir, and extracting the oil from the reservoir.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional of and claims priority to U.S.Provisional Patent Application Ser. No. 61/243,472, filed Sep. 17, 2009,which document is hereby incorporated by reference herein to the extentpermitted by law.

BACKGROUND OF THE INVENTION

Approximately sixty-five percent of all the oil discovered remainstrapped underground in reservoirs following primary production (naturalreservoir pressure) and secondary production (water or gas flood).Microbial enhanced oil recovery (“MEOR”) holds considerable promise forrecovering a significant proportion of trapped global oil reserves.

Conventional MEOR is an empirical process whereby inexpensive nutrientsare pumped into an oil reservoir to stimulate growth of indigenous anddormant microorganisms. In theory, the rejuvenated microbial communityproduces environmentally friendly biometabolites such as gases, acids,solvents, and surfactants that release trapped oil and/or biomass andpolymers that plug water channels thereby diverting subsequent water orgas floods into oil bearing zones.

Conventional MEOR has been employed for decades and has been moderatelysuccessful but, frequently, the results have been disappointing. Atypical MEOR approach is to pump molasses or agricultural fertilizerinto a watered-out reservoir and hope for the best. This hit-or-missapproach is not based on scientific principles and any positive,negative, or damaging results remain unexplained. In some cases,undesirable bio-metabolites such as hydrogen sulfide have causedirreversible reservoir damage, equipment corrosion, and health threats.

There are many applications of MEOR, but none of them include priormetabolic characterization of microbial communities that inhabit oilreservoirs. According to some culture-based and genetic evidence,microbial communities are markedly different among oil reservoirsdepending on rock type, temperature, depth, and various other factors.Therefore, blindly injecting nutrients into an oil reservoir and hopingfor beneficial results is an uncertain and potentially damaging process.Pumping the same nutrient into several reservoirs and expecting similarresults is unscientific and unreasonable. There is no way currently topredict what bio-metabolic response, if any, can be expected in a givenoil reservoir when nutrients are injected. Therefore, it would bebeneficial to have a method for growing reservoir microorganisms in acontrolled and scientific way.

Targeted, scientifically-based MEOR treatments could be devised forindividual oil reservoirs if one knew the likely metabolic response ofthe microbial community to an infusion of nutrients. Then one would needto stimulate the desirable microbes and suppress the undesirable ones,for example, sulfate-reducing bacteria responsible for souring oil. Todo this in a scientific fashion, one has to know what species ofbacteria live in a given reservoir, what the actions of the microbialcommunity in a given reservoir to nutrient infusions, what bioproductsthey are capable of producing, and exactly what nutrients and co-factorsthey need to grow at optimum rates. However, most reservoir microbes diewhen brought to the surface in a sampler, when those microbes areexposed to air, low temperature, low pressure, and a variety of otherstressors. Few, if any, indigenous microbial species survive whenhoisted to the surface. Therefore, conventional laboratory culture ofoil-reservoir microorganisms in Petri dishes or in flasks of liquidgrowth media at room temperature is not feasible.

The basic goals of MEOR are: (1) to stimulate desirable reservoirmicrobes, for example, those that produce useful quantities ofoil-releasing or channel-plugging materials; and (2) to suppressundesirable ones, for example, sulfate-reducing bacteria responsible forsouring oil, corroding pipes and equipment, and that pose a toxic hazardto workers. To do this in a scientific and predictive fashion, one mustknow what bio-metabolites reservoir microbes are capable of producing,and exactly what nutrients, supplements, and co-factors they need togrow and produce specific bio-metabolites at optimum rates. However,culturing reservoir microorganisms in the laboratory to elucidatemicrobial community metabolism is unsatisfactory because ninety-ninepercent or more die when brought to the surface because of the suddendecreases in pressure and temperature and the exposure to oxygen.Therefore, it would be beneficial to have a method for growing reservoirmicroorganisms under anaerobic conditions and under reservoir conditionsof high temperature and high pressure.

SUMMARY OF THE INVENTION

The present invention is generally directed to a method for releasingtrapped oil in reservoirs. The method hereof includes identifying areservoir, obtaining a microbial community sample of the reservoir,maintaining the sample under high temperature and high pressure (“HTHP”)conditions that mimic natural conditions of the reservoir, growing thesample on at least one substrate, determining a targeted treatmentregime for the reservoir based on the positive growth of the sample onthe substrate, injecting the reservoir with the targeted substrate torelease trapped oil in the reservoir, monitoring the reservoir, andextracting the oil from the reservoir.

Since reservoir microbes exist under conditions of high temperature andhigh pressure in an anoxic and usually hypersaline environment, whensampled and brought to the surface, exposure to oxygen, lowtemperatures, low pressures, and lower salinity kills virtually all ofthe microbes. If one were able to culture them in different nutrientgrowth media under reservoir conditions, i.e., in a HTHP chamber,accurate measurements of metabolic by-products could be made.

Under HTHP culture, the by-products of microbes from a specific oilreservoir could be identified and predictions of growth and metabolismof the microbial consortium in the presence of a given nutrient mixcould be obtained. By culturing the consortium in a number of nutrientgrowth media and chemically and physically measuring acids, gases,solvents, surfactants, biomass, and polymers produced, predictions couldbe made about specific metabolic by-products to be expected in a givenoil reservoir when injected with a specific nutrient medium.

If, for example, bacterial biomass and polymers are needed to blockwater channels, if acid production is required to dissolve carbonaterock, if gas production is needed to repressurize the reservoir, and ifnitrates are needed to suppress sulfate-reducing (oil-souring),bacteria, a treatment regime (perhaps sequential) could be devised for aspecific reservoir based on the HTHP chamber culture results.

Other and further objects of the invention, together with the featuresof novelty appurtenant thereto, will appear in the course of thefollowing description.

DETAILED DESCRIPTION OF THE INVENTION

There is provided herein an integrated method that significantlyenhances the recovery of crude oil. The method of the present inventiongenerally includes: (a) growing reservoir microorganisms ex situ in avariety of growth media under high temperature/high pressure (“HTHP”)conditions that mimic reservoir conditions; (b) characterizing theirmetabolic byproducts; and (c) injecting a tailored nutrient medium intothe reservoir that releases trapped oil via a variety of potentialmechanisms.

The method of this invention exploits a novel application of MEOR.Specifically, the method of the present invention is a process wherebyliving microorganisms are obtained from oil reservoirs, are maintainedat bottom hole temperatures and pressures, and are grown on differentnutrient substrates in a high temperature/high pressure growth chamber.Microbial aqueous and gaseous metabolic by-products are measured bychemical and physical assays during and following the microbial growthphase and the results are used to develop specific reservoir treatmentregimes with nutrients to stimulate desirable microbes, to suppressundesirable microbes, and to insure maximum production of microbialmetabolic by-products that stimulate enhanced production of trapped oil.Essentially, reservoir microbes are cultured under reservoir conditions,the microbes' metabolic by-products are measured, and specific treatmentregimes are developed for individual reservoirs to release trapped oil.

During the productive life of a typical oil well, the first ten percentof oil flows spontaneously to the surface because of undergroundpressure in the reservoir. This process is called primary production.Another twenty-five percent of the original oil in place (“OOIP”) canusually be produced by secondary treatment of the reservoir. Thistypically involves injecting the reservoir with water under highpressure, forcing the oil ahead of the waterflood from an injector wellto producer wells. However, water usually finds the path of leastresistance through the rock substrate of the reservoir, and eventuallywater channels are formed directly from the injector well to producerwells. The amount of water appearing in the oil at the producer well iscalled the “water cut.” Oil wells are usually capped and abandoned whenwater cuts exceed ninety to ninety-five percent, leaving most of theOOIP behind. The amount of trapped oil in the United States alone isestimated at 377 billion barrels.

Light, sweet crude oil reservoirs that are not too hot, too saline, orunder too much pressure usually host a diverse community ofmicroorganisms. The majority of these live at the oil-water interface(the residual oil zone or “ROZ”), and are basically in a state ofsuspended animation, having used up available nutrients over time andhaving produced growth-limiting metabolic wastes. An infusion of propernutrients stimulates these microorganisms to grow. Table 1 hereinbelowillustrates a variety of products potentially useful in oil recovery andthe typical effect those products have.

TABLE 1 Bioproduct Effect Acids Modification of reservoir rock Improvedporosity and permeability Reaction with calcareous rocks a CO₂production Biomass Selective or nonselective plugging Emulsification viaadherence to hydrocarbons Modification of solid surfaces (biofilms)Degradation or alteration of oil Reduction of oil viscosity and oil pourpoint Desulfurization of oil Gases Reservoir repressurization Oilswelling Viscosity reduction of oil Increased permeability (CO₂solubilization of carbonate rocks) Solvents Reduce oil viscositySurfactants Lowered interfacial tension Emulsification Polymers Mobilitycontrol Selective or non-selective plugging

In one embodiment, the acids may be, but are not limited to, formicacid, acetic acid or valeric acid. In another embodiment, the gases maybe, but are not limited to, carbon dioxide, methane, hydrogen sulfide,or hydrogen. In one embodiment, the solvents are primarily alcoholsincluding, but not limited to, methanol, ethanol, propanol, isobutanol,and n-butanol. In another embodiment, the solvents are formaldehyde andacetone. In yet another embodiment, the solvents are aldehydes orketones. In another embodiment, surfactants may be, but are not limitedto, anionic compounds such as, for example, carboxylic acids; cationiccompounds such as, for example, amines and heterocyclic compounds;amphoteric compounds such as, for example, amino acids and peptides;non-ionic compounds such as, for example, esters; and poly-anioniclipids. In yet another embodiment, the polymers may be, but are notlimited to, proteins and polysaccharides.

One embodiment of the method of the present invention provides aninnovative method (i.e. anaerobic culture of microbes in reservoirfluids at subsurface conditions of temperature and pressure) to predictprecisely what bio-metabolites will be produced in a targeted reservoirfrom treatment with various nutrient media. Based on these results and areview of the geology of the reservoir, a precise nutrient-mediumformulation is devised and injected into the reservoir in optimumquantities to (a) plug watered-out channels, (b) to induce maximumproduction of other desirable biometabolites, and/or (c) to suppressgrowth and metabolism of undesirable microbes, especially those thatproduce hydrogen sulfide and sour oil. At least one result is theenhanced recovery of oil. An objective of the present invention is toincrease the amount of oil ultimately produced above and beyond whatwould have been recovered using other treatments. This depends mainly onincreased displacement of oil and/or improved volumetric efficiency offlooding techniques.

A reservoir is screened to determine if the reservoir is a candidate foruse with the MEOR method of the present invention. Generally, indicatorsthat a reservoir is a good candidate for use with the MEOR method of thepresent invention include, but are not limited to, salinity (NaClcontent) of the formation water less than about 10% wt/vol; temperatureless than 167-180° F.; depth of reservoir less than 8,000 feet; presenceof trace elements As, Se, Ni, and Hg, less than 10-15 ppm. Permeabilitygreater than approximately 50 mD to 75 mD; oil gravity greater than15°-18° API; residual oil saturation greater than 25%; and/or pH between4 and 9. Another indicator is that the concentrations of cations andanions at certain levels might interfere with microbial systems. Thosecations and anions and their respective concentrations are approximatelylithium 400 mg/l, barium 670 mg/l, boron 450 mg/l, bromine 6,000 mg/l,and iodine 1,400 mg/l.

Once biometabolities are elucidated via HTHP growth in the laboratory, aspecific treatment regime would be developed for that reservoir. In oneembodiment the treatment regime includes, but is not limited to, (1)developing an ideal formulation of nutrients, salts, growth factors, andsuppressants; (2) injecting nutrients with initial waterflooding orwaiting until later in the cycle; (3) injecting nutrients followingfishbone drilling to reach out-of-the-way pockets of oil; (4) sequentialinjections (e.g., first to create biomass to plug thief, i.e.,watered-out, zones, then nutrients to stimulate oil production or tochannel CO₂ in an enhanced miscible CO₂ flood).

Some advantages of targeted MEOR over other previous recovery techniquesare: (a) potential low costs (chemical tertiary methods such as polymerfloods, surfactant floods, and others are expensive); (b) multiplemechanisms working simultaneously thereby enhancing effectiveness; (c)environmentally benign; (d) exploiting indigenous reservoirmicroorganisms requires only nutrient infusions similar to conventionalwaterfloods; (e) ability to complement and enhance selected secondaryand tertiary recovery techniques including waterfloods and miscible CO₂floods; and (f) prior characterization of reservoir response assurespredictable and reliable MEOR results.

In one embodiment, once a target reservoir is selected, bottomholesamples are obtained, and the samples are maintained at reservoirtemperatures and pressures during sampling, during transport to thelaboratory, and during culture. Reservoir microbes are grown in a seriesof candidate nutrient media in HTHP anaerobic chambers that mimicreservoir temperature and pressure, i.e., reservoir simulators. Bymeasuring biomass and by chemically analyzing bio-metabolites producedin the laboratory, one obtains accurate data to guide nutrientselection, concentration, and time for a targeted reservoir, therebyinsuring maximum release of trapped oil and mitigating risk of reservoirdamage.

A conventional industry PVT-type sampler may be used to obtainbottomhole samples, usually bringing two each 600-ml fluid samples tothe surface. The reservoir samples are obtained from the oil/waterinterface (residual oil zone). Additional samples can be obtained ifrequired. The bottomhole temperature and pressure are measured by thesampler itself or by a separate probe. Pressure in the canisters ismaintained at bottomhole pressure by hydraulics or nitrogen injection ascanisters are retrieved to the surface. Bottomhole samplers aremanufactured by a variety of different oilfield service companies, andreservoir sampling support can be contracted.

“Slickline” sampling can retrieve canisters at 200 feet/minute. Usingthis sampling scheme, pressure is maintained, but temperature is notduring ascent of the sampler up the well casing. However, sample transitvia slickline from 4,000 feet is only 20 minutes, the sampling canistersprovide good insulation, and subsurface microbes are probably not assusceptible to a temperature drop of a few degrees for a short time asthey would be to a precipitous drop in pressure. Samples are transferredunder pressure to sample-transport canisters, and transport canisterscontaining pressure-compensated samples are placed into a portable ovento maintain bottomhole temperature during transport to the laboratory.

Prior to transfer to HTHP growth chambers, reservoir samples are storedin an oven to maintain reservoir temperature. Reservoir bottomholepressure is maintained inside the sample bottles, i.e., they are notopened prior to fluid transfer.

In order to culture reservoir microorganisms under reservoir conditions,any of several HTHP growth chambers are suitable. These chambers areused for growing reservoir microbial consortia in various growth media,including modifications of in situ growth chambers designed for studyingdeep-sea hydrothermal vents. In one embodiment, reservoir simulator isused, and it provides for transfer of samples under pressure, for growthand metabolism under reservoir conditions, and for periodic monitoringand analysis of headspace gas, real-time monitoring of pH, and periodicremoval of liquids for physical (e.g. biomass) and chemical analyses. Inaddition, a piston arrangement allows for pressure compensation in theupper growth chamber as metabolic gases are liberated during microbialgrowth. Growth chambers are usually assembled in a manifold array toprovide for serial dilutions of reservoir oil/water inoculums into aselected nutrient medium if required, or to provide for inoculationsinto several types of nutrient media. Bottomhole reservoir samplebottles containing remaining reservoir fluids are maintained atreservoir temperature and pressure between inoculations.

One embodiment and a non-limiting illustrative example of the method ofthe present invention is outlined below and includes the followingsteps: (1) An oil company identifies and characterizes light, sweetcrude oil reservoir that matches preliminary MEOR screening criteria, asdisclosed herein. (2) A contracted petroleum laboratory conductsconventional PVT fluid-property and other analyses on sample ofretrieved oil (these data are probably already available). (3) An oilcompany obtains two 600-ml reservoir samples from oil/water interface(residual oil zone or ROZ). Both canisters are maintained at bottomholepressure by nitrogen injection as canisters are retrieved to thesurface. “Slickline” sampling can retrieve canisters at 200 feet/minute.(4) Canisters containing pressure-compensated samples are placed into“pizza oven” device for transport. Oil-service companies will becontacted to determine if canisters can be insulated to preventsignificant temperature drop during transit from bottomhole up thecasing to the surface. (5) One canister is placed into hightemperature/high pressure (HTHP) robotic chamber for subsequent opening,inoculation, and microbial growth on various substrates (specificprotocol to be determined). (6) The sample from the second canister issplit. Approximately one-half is used for room-temperature cultureattempts for indigenous microbes that will survive at ambienttemperature and pressure. The other one-half is for archiving forsubsequent metagenomic sequencing and/or culturing of desirablemicroorganisms. (7) Growth and metabolic by-product studies areconducted of bottomhole microbial consortium in various liquid growthmedia including molasses, nitrogen/phosphate fertilizers, and varioustreatment grades of industrial wastes such as paper/pulp, sugar beet,brewery, and feedlot. (8) The growth of microbial consortia in varioustypes are assessed and dilutions of growth media by measuring (a) changein turbidity of growth medium, (b) numbers of microbes per ml (i.e.,biomass), (c) volume of headspace gases produced, and (d) other measuresof growth. (9) Samples of headspace gases and liquid culture medium areobtained for (a) chemical and volumetric analyses of headspace gases and(b) chemical nature of metabolic byproducts in the growth medium frommicrobial growth such as pH change, surfactants produced, polymersproduced, and solvents produced. Alternatively, a battery ofpetroleum-lab screening tests in-house for “black-box” efficacy studiesof fermented liquid media in releasing trapped oil may be used. Thesetests could include Berea cores and sand packs, surfactant break tests,and many others.

Based on measurements of microbial growth, samples from media dilutionsexhibiting significant growth will be gathered and will be filtered andfrozen for subsequent metagenomic analysis and for subsequent growthexperiments at ambient temperature and pressure.

Fundamental data on reservoir fluids should be available from the welloperator, e.g. PVT, API gravity, and other fluids data, as well asreservoir characteristics such as depth, porosity, permeability,residual oil saturation, API, water cut, and other information. Rawreservoir samples are analyzed in the laboratory for pH, biomass, anddissolved gases. In addition, chemical analyses are used to characterizeoil samples as described in a chemistry discussion below.

Growth chambers are loaded with selected sterilized nutrient media atambient temperature and pressure, and then the chambers are closed,charged with nitrogen or inert gas, and brought up to reservoirtemperature and pressure. A tubular connection with pressure gaugeenables transfer of a portion of reservoir fluid (inoculum) to theloaded HTHP growth chamber. During transfer of reservoir fluids, thechamber pressure is maintained at slightly less than the reservoirsample bottle to provide for metered fluid flow into the growth chamberthat is not too turbulent, but is sufficiently turbulent to mixreservoir fluids and nutrient media thoroughly.

Many types of growth media are suitable for use including thosetypically used in empirical MEOR applications in the field. ConventionalMEOR solutions include but are not limited to: molasses (an inexpensivecarbon source with micronutrients that is commonly used in MEOR), 0.5%aqueous solution (vol/vol) more or less; augmented molasses: 0.5%molasses, 0.15% KNO3 (w/v), and 0.05% Na3PO4 (w/v), or variationsthereof; or an aqueous solution of fertilizer: 0.25% KNO3 (w/v), and0.05% NaH2PO4 (w/v), or variations thereof.

Many other types, concentrations, and mixtures of growth media aresuitable for trials in the HTHP growth chambers. Regional industrialwaste streams are evaluated to determine their suitability as potentialnutrient media for MEOR. The fundamental appeal of such a “green”approach is that local waste streams could be diverted to productive andvery profitable use for enhanced oil recovery. Because of regulatoryrequirements, many industrial waste streams have already beencharacterized by the supplier, and these chemical data sheets are usedas background. Candidate aqueous wastes include those from breweries,food processing plants, sugar (beet and cane) refineries, pulp and papermanufacture, animal feedlots, treated municipal wastes, and many others.Industrial wastes are generally pre-filtered to remove suspended solids.Aliquots of nutrient media formulations and waste streams are analyzedchemically as described below before inoculation and incubation in HTHPgrowth chambers.

Typically, ninety milliliters each of MEOR nutrient solutions andindustrial waste streams are loaded into separate HTHP chambers underambient pressure, and the vessels are then charged with inert gas andpressurized. Ten milliliters of reservoir fluids from a single well arethen added to each of the HTHP growth chambers under reservoir pressure,i.e. a 10% inoculum. Larger or smaller HTHP growth chambers can be usedand inoculum ratios can be modified depending on requirements and growthresponses.

During incubation, growth-chamber pressure is maintained at in situreservoir pressure by hydraulic or electrical (screw) manipulation of apiston. Growth chambers are immersed in a water bath or are subject toan alternative heating method to maintain reservoir temperature. Theentire apparatus is generally moved to a fume hood for containment. Allchamber sensors can be connected to a control computer for process pH,gas-generation, and other monitoring and data acquisition.

Measurements of acid, gas, and biomass production is disclosed herein.Typical incubations are expected to take approximately 2-6 weeks each,and the end point is determined by cessation of acid and gas production.The volume and composition of metabolic off gases and pH of the nutrientmedium are analyzed periodically in samples removed from the growthchamber to obtain gas-generation (via gas chromatograph) andacid-generation (via pH meter) curves for each reservoir-nutrientcombination. Sensors can be incorporated into the construction of theHTHP growth chamber to monitor pH, pressure, gases, and other parametersand constituents remotely and in real time. Biomass is calculated duringand at the end of incubation by cell count, turbidity, filtering andweighing, and/or other measurements to obtain microbial growth curves.

Following incubation, liquid samples are transferred to a chemicallaboratory for analysis. Chambers are cleaned and sterilized usingacceptable methods. The growth chambers can be disassembled, cleanedwith a solvent to remove hydrocarbon residues, and thenautoclave-sterilized at 121° C. or equivalent.

Chemical analyses of reservoir fluids and growth media are disclosedherein. Representative samples of reservoir fluids, uninoculatednutrient solutions/suspensions, and growth media following incubationfrom microbial reactions are analyzed chemically. As described below,the easiest and probably most profitable application of MEOR is pluggingof water channels (fingers or “thief zones”) in watered-out reservoirsvia biomass and/or bio-polymer produced by nutrient-stimulated microbesin situ. For that reason, a high priority is given initially todeveloping, modifying, refining, and applying simple, straightforwardmeasurement techniques for production of biomass and bio-polymers inreacted HTHP chamber samples.

Analytical data from chemical analyses would be difficult to interpretexcept in the context of changes observed in samples before and aftermicrobiological action. For example, a nutrient source that isrelatively high in sulfur content could produce an undesirable outcomefrom in situ microbial activity in a reservoir versus a source that islower in sulfur. Also, some of the target microbial bio-metabolitesmight already exist in the reservoir fluids, and could not necessarilybe shown to result solely from the laboratory incubation process.Chemistry data from the unreacted reservoir fluid and the uninoculatednutrient solutions are therefore necessary to interpret the results ofthe tests.

Representative samples from each source of reservoir fluid employed forHTHP growth are chemically analyzed before inoculation. These samplesinclude water or other residual materials used in previous recoveryefforts from the well(s), and it is assumed that samples contain bothaqueous and non-aqueous phases. While the total volume and proportion ofaqueous and non-aqueous phases vary from reservoir to reservoir,standard methods for sample preparation are utilized. These includecentrifugation, filtration, and fractionation, where possible.

Representative samples of each type of nutrient source are analyzed bymethods selected from those outlined below. These differ from those usedto analyze the control reservoir fluid and the organic phase of theincubated media, and focus on certain nutrients and incidentalconstituents, such as proteins, peptides, alcohols, organic acids,sugars, N, P, K, Na, and S. Any samples that are transferred betweenlaboratories are shipped through commercial means under refrigeration inorder to minimize ongoing microbiological activity during storage andtransit.

The incubated nutrient media from tests often requires additionalpreparation and pre-separation of phases prior to chemical analyses. Themethods used for these samples are designed to obtain useful data fromany separable organic and aqueous phases over the full range ofapplicable analytes.

The analyses selected for each of the matrices listed are based on thetypes of data needed to evaluate the results of media testing, includingdiagnostic differences between incubation runs and nutrient mixturesused. Most analyses follow specific pre-separations or other preparationsteps. The target analytes may include but not be limited to: petroleumconstituents of different classes, molecular weight, polarity, andvolatility (analyses focuses on profiles, peak patterns, and relativedifferences between samples, rather than specific identification orquantification of individual constituents); surfactants (e.g.,carboxylic acids, amines, amino acids, peptides, esters); solvents(lower molecular weight constituents that would dilute and decreaseviscosity of oil, e.g., alcohols, ketones, and aldehydes);polysaccharides (could be introduced in the nutrient mixture, orproduced as metabolites and capsular polysaccharides of bacteria in theincubated media); proteins (include soluble proteins and membraneproteins, possibly originating in the nutrient mixtures or frommicrobial growth); organic acids (survey of lower molecular weightorganic acids, e.g., formic, acetic, and valeric); and/or specificmineral nutrients and inorganic cations (elemental analysis of N, P, K,Na, S).

Methods generally selected from, but not limited to, the following areused in analysis of samples for the types of analytes listed above.Chemical analyses provide sufficient data on types and quantities ofanalytes produced to make informed decisions on what nutrient mediumproduces best bio-metabolite results for the reservoir in question. Allwork is performed according to accepted standards of documentation andquality assurance. The final selection of methods is partially dependenton the kind, volume, and number of samples.

The qualitative analysis of both known and unknown non-volatilechemicals can be performed using liquid chromatography/mass spectrometry(LC/MS). Samples such as the organic and aqueous phases of unreactedreservoir fluid, unreacted nutrient solutions, and organic and aqueousphases of incubated media are prepared for analysis using suitablepre-separation and extraction methods, and are then introduced into theLC/MS system. The system allows for the chromatographic separation ofchemicals in the sample such as acids, solvents and surfactants prior tointroduction into the mass spectrometer. Because the chemicals ofinterest can be somewhat separated prior to introduction into the massspectrometer, a total ion fingerprint can be obtained by full scananalysis with the mass spectrometer.

Identification information from the fingerprint region can be obtainedfrom individual peaks in the chromatogram to determine the number ofcompounds in each peak as well as information on potential molecularions of individual chemicals. Additional testing, if warranted, includesproduct ion scans (LC-MS/MS) of each separated chemical in which themolecular ion is isolated and fragmented (utilizing collision induceddissociation). The product ions (fragments), as well as the calculatedneutral losses, are then used to propose structures or chemical classesfor the constituents extracted from the original samples.

Gas chromatography/mass spectrometry (GC/MS) is an additional tool thatis used, as appropriate, in the analysis of the various samples. It isparticularly well-suited for the identification of petroleumconstituents such as alkanes, alefins, naphthenes, and aromatics, aswell as solvents that may be present in these samples. Initially,full-scan GC/MS is used to analyze samples that have been suitablyextracted and prepared. As in the LC/MS analysis, these data are used toobtain an overall picture of a sample (e.g. number and types ofcompounds present), which can then be followed by more targetedanalyses. Mass spectrometry techniques and instrumentation for in-depthanalysis of samples include, but are not limited to the followingsamples outlined below.

A High Resolution MS (e.g. Waters AutoSpec Premier) instrument capableof high R>10,000 amu mass spectral resolution to improve detectionconfidence in the presence of interfering ions.

Heart-Cut GC-GC/MS systems consisting of gas chromatographs coupled witha mass spectral detector and equipped with heart-cutting systemsoffering excellent matrix rejection through the selective “cutting” ofdiscrete effluent from the first C column and placement on the secondcolumn.

Multi-Dimensional systems, e.g. GC×GC-TOFMS (Leco GC×GC-TOFMS, Pegasus4D) capable of performing multi-dimensional GC separations throughthermal modulation GC×GC. Such separations provide an alternative,multi-dimensional separation versus heart-cutting through an advancedthermal modulation system and are increasingly being used for theanalysis of petroleum samples. The technique enables the increasedseparation of alkanes, olefins, naphthenes, and aromatics with limitedsample preparation making it well suited to unknown analyses.

A variety of detectors, other than mass spectrometry, can also be usedfor coupling with gas chromatography for sample analysis, includingPulse Flame Photometric Detectors, Flame Ionization Detectors, andElectron Capture Detectors.

Due to partition of some analytes between aqueous and non-aqueousphases, some of the same materials can be observed by both GC/MS andLS/MS depending on the pre-separation and preparation methods selected.

Proteins and other larger molecular compounds are qualitatively analyzedusing Matrix-Assisted Laser Desorption/lonization Time-of-Flight MassSpectrometry (MALDI-TOF MS) or LC/MS/MS technologies. Samples areprocessed using suitable extraction methods (e.g., filtration,centrifugation, molecular weight cutoff separation, chromatography, orprotein digestion) and analyzed by MALDI-TOF MS or LC/MS. MALDI-TOF MSallows for detection of intact high molecular-weight compounds,including peptides and proteins, up to 100-200 kDa. Limited compoundseparation is typically performed prior to analysis, resulting in amolecular fingerprint of the hundreds of compounds detected from thecomplex sample milieu. Proteins can also be purified from the samples,digested and analyzed by LC/MS, allowing for detection of the sample'sunique proteomic fingerprint. Protein identification, if warranted, canbe performed using tandem MS (MS/MS) techniques in which individualpeptides are isolated and fragmented, for further samplecharacterization.

In addition to the kinds of sample constituents that would be analyzedby either GC/MS or LC/MS, there are a number of other constituents fromeach of the sample matrices that could require either wet chemistry orspecialized instrumental methods. Some of the following might beapplicable: conventional elemental analysis, ICP/MS, or AA; nutrientelements (N, P, K, Na, S); trace elements (As, Se, Ni, and Hg); elementsinterfering with microbial growth (Li, Ba, B, Br, I); neutron activationanalysis—total element surveys, and/or semiquantitative determination ofspecific elements, in isolated fractions (inexpensive fingerprint ofelemental makeup); Karl Fischer—water analysis; optical or NMRspectroscopy—Analysis of specific isolated and recovered organicconstituents (if needed); and/or physical constants (less likely)—e.g.,melting point, boiling point, optical rotation, density

The results of these or, more likely, a smaller subset of these physicaland chemical analyses will enable operators to predict reservoir effectsof specific nutrient solutions injected into a reservoir for MEORpurposes. As the technology is adopted in declining and spent oil fieldsaround the globe, chemical analytical methods and other measurementtechniques will likely be refined and focused to provide maximum dataquickly and at reduced costs. For example, if channel plugging is theprimary goal in a particular watered-out reservoir, a series of quicktests for biomass and polymer/slime formation could evolve to providethe requisite information rapidly and inexpensively.

Some models predict that in situ MEOR production of surfactants or gasesis likely to have little or no effect on enhancing oil recovery.Therefore, careful laboratory HTHP studies of actual bio-productsproduced by reservoir microorganisms in various nutrients and theirability to release trapped oil using a variety of petroleum-laboratorytests are warranted before large-scale reservoir nutrient injections areattempted. Promising bio-metabolites are tested in the laboratory usingconventional industry test beds (e.g. surface tension tests, sand packs,and core floods) to quantify oil-release and channel-plugging potentialbefore conducting field tests on selected oil reservoirs. To obtainsufficient quantities of bio-metabolites for these tests, larger HTHPgrowth chambers may be used.

Desirable reservoir microorganisms that can be cultured ex situ atambient temperature and pressure are isolated in the laboratory frombottomhole samples. These are evaluated for possible future reservoirinjection. Reservoir microbial communities are characterized via 16Sribosomal RNA gene sequencing, metagenomic sequencing, andbioinformatics analysis; results are used to significantly improvenutrient selection and to enhance MEOR processes over the long term.

The products of this novel process described herein include: (a) anannotated list of biometabolites produced by microorganisms in atargeted reservoir grown under reservoir conditions in a number ofcandidate nutrient media; (b) detailed chemical characterization ofbiometabolites; (c) results of culturing reservoir microorganisms underambient conditions to isolate indigenous microbes that can be grown infermenters and reinjected into the reservoir to further enhance oilrelease; (d) results of metagenomic studies and bioinformatics analysisto improve the process; (e) results of petroleum-laboratory tests toestimate channel-plugging and/or oil-releasing efficacy of variousbio-metabolites; and (f) injection of optimum amounts of nutrientformulations at optimum concentrations and for optimum times in targetedreservoirs to enhance oil recovery and to avoid or to suppress souringand to avoid other undesirable or damaging effects to the reservoir,equipment, public health, and the environment.

In concert with reservoir engineers, the sub-surface volume ofwatered-out channels is calculated, and optimum amounts andconcentrations of nutrients are injected into the reservoir to plugfingers, i.e. “thief zones”. The well is capped for a period of timedetermined, in part, by results of HTHP experiments, and then secondaryfloods with water and/or CO₂, or other gases, or other treatments areinitiated or resumed.

Results of petroleum-laboratory tests in sand packs, cores, etc. usingactual bio-metabolites are used to estimate potential reservoir efficacyof various nutrient treatments. Sequential reservoir treatments areemployed if indicated; e.g. MEOR-induced channel-plugging followed bynutrient injections to reach previously bypassed oil-bearing zones.Follow-up nutrient injections are targeted at producing desiredbio-metabolites such as acids, gases, solvents, and surfactants asdetermined by petroleum geologists and reservoir engineers for thespecific reservoir.

The described process technology may only release trapped oil, but canalso be used to study any underground microbial community where pressureand temperature have to be maintained in the laboratory. Other importantpotential applications include, but are not limited to: biodegradinglarge, heavy-oil molecules in order to lower viscosity and improveproduction; In situ bio-upgrading of heavy oil and oil sands deposits;bio-converting trapped or uneconomical hydrocarbon deposits into moreeasily produced methane; and Uranium bioleaching in underground deposits

If, for example, bacterial biomass and polymers are needed to blockwater channels, if acid production is required to dissolve carbonaterock, and if nitrates are needed to suppress sulfate-reducing(oil-souring) bacteria in a specific reservoir, treatment regimes can becustom-designed based on the HTHP-chamber culture results, and sandpack/core verification tests.

This invention will provide scientifically derived information directlyapplicable to target oil reservoirs that will enable operators torecover trapped oil, i.e. oil that cannot be recovered by conventionalprimary and secondary recovery techniques.

Additional applications for the method of the present invention are (a)CO₂ or water-after-gas CO₂ floods following channel plugging; (b)producing acid in carbonate reservoirs to dissolve reservoir rockthereby increasing permeability and porosity; (c) co-producing solventsand surfactants to lower interfacial tension and reduce oil viscosity;and (d) production of CO₂ and other gases to repressurize reservoirs,and to cause viscosity reduction and swelling of oil.

Additional expansion of the applications include, but are not limited to(a) MEOR for heavy oil deposits; (b) biodegrading large, heavy-oilmolecules in order to lower viscosity and improve production; (c) insitu bio-upgrading of heavy oil and oil sands deposits; and (d)bio-converting trapped or uneconomical hydrocarbon deposits into moreeasily produced methane.

From the foregoing it will be seen that this invention is one welladapted to attain all ends and objects hereinabove set forth togetherwith the other advantages which are obvious and which are inherent tothe structure. It will be understood that certain features andsubcombinations are of utility and may be employed without reference toother features and subcombinations. This is contemplated by and iswithin the scope of the claims. Since many possible embodiments may bemade of the invention without departing from the scope thereof, it is tobe understood that all matter herein set forth is to be interpreted asillustrative, and not in a limiting sense.

1. A method for growing and metabolizing microbes comprising the stepsof: identifying a reservoir; obtaining a microbial community sample ofsaid reservoir; maintaining said sample under high temperature and highpressure conditions that substantially mimic natural conditions of saidreservoir; growing said sample on at least one substrate; determining atargeted treatment regime for said reservoir based on positive growth ofsaid sample on said substrate; injecting said reservoir with saidsubstrate to release trapped oil in said reservoir; monitoring saidreservoir; and extracting said oil from said reservoir.
 2. The method ofclaim 1 further comprising the step of reinjecting said reservoir withsaid substrate.
 3. The method of claim 1 wherein said determining stepfurther comprises the step of developing a substantially ideal treatmentformulation including compounds selected from the group consisting ofnutrients, salts, growth factors, suppressants, and combinationsthereof.
 4. The method of claim 1 wherein said injecting step furthercomprises injecting said nutrient via fishbone drilling.
 5. The methodof claim 1 wherein said substrate is molasses.
 6. The method of claim 1wherein said substrate includes at least one targeted nutrient.
 7. Themethod of claim 6 wherein said nutrient is selected from the groupconsisting of acids, biomass, gases, solvents, surfactants, polymers,and mixtures thereof.
 8. The method of claim 7 wherein said solvents areselected from the group consisting of primary alcohols, acetone,aldehydes, ketones, and mixtures thereof.
 9. The method of claim 7wherein said surfactants are selected from the group consisting ofcarboxylic acids, amines, heterocyclic compounds, amino acids, peptides,esters, poly-anionic lipids, and mixtures thereof.
 10. The method ofclaim 7 wherein said gas is selected from the group consisting of carbondioxide, methane, hydrogen, hydrogen sulfide, and mixtures thereof. 11.A method for releasing trapped oil in a reservoir comprising the stepsof: identifying a reservoir; obtaining a sample of a microbial communityof said reservoir; isolating said sample under high temperature and highpressure conditions that substantially mimic natural conditions of saidreservoir; testing said sample under said high temperature and highpressure conditions; developing a targeted treatment regime based ontest results produced from said testing, wherein said targeted treatmentregime includes at least one target substrate; performing said treatmentregime; and extracting said oil from said reservoir.
 12. The method ofclaim 11 wherein said targeted treatment regime further includesdeveloping a substantially ideal formulation of nutrients, salts, growthfactors, suppressants, and combinations thereof.
 13. The method of claim11 wherein said target substrate is molasses.
 14. The method of claim 11wherein said target substrate includes at least one targeted nutrient.15. The method of claim 14 wherein said nutrient is selected from thegroup consisting of acids, biomass, gases, solvents, surfactants,polymers, and mixtures thereof.
 16. The method of claim 15 wherein saidsolvents are selected from the group consisting of primary alcohols,acetone, aldehydes, ketones, and mixtures thereof.
 17. The method ofclaim 15 wherein said surfactants are selected from the group consistingof carboxylic acids, amines, heterocyclic compounds, amino acids,peptides, esters, poly-anionic lipids, and mixtures thereof.
 18. Themethod of claim 15 wherein said gas is selected from the groupconsisting of carbon dioxide, methane, hydrogen, hydrogen sulfide andmixtures thereof.